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Minireview 223

Firefly : the is known, but the mystery remains Thomas O Baldwin

The structure of luciferase reveals a new protein its starting condition. , on the other hand, fold which may be representative of a growing family of catalyzes an oxidative reaction involving ATP, firefly homologous . and molecular , yielding an electronically excited oxyluciferin [2]. This excited species Address: Department of Biochemistry and Biophysics and emits visible , which is employed by the firefly in its Department of Chemistry, Center for Macromolecular Design, reproductive behavior [3]. Firefly luciferase was one of the Texas A&M University, College Station, TX 77843–2128, USA. first enzymes to be investigated in biochemical detail [4]. Structure 15 March 1996, 4:223–228 WD McElroy and his colleagues [5–9], as well as other investigators [10] working during the 1940s and 1950s © Current Biology Ltd ISSN 0969-2126 with P. pyralis, determined the of the substrates and products of the . Evidence for the chemical From the Atlantic Ocean to the eastern face of the Rocky mechanism proposed for the firefly luciferase reaction [11] Mountains, a summer sunset is usually accompanied by has been discussed in a recent review [12]. one of Nature’s most delightful light shows. , the North American firefly, has entertained count- The structures of less observers, probably since the coming of humans to The structure of luciferase from the firefly P. pyralis has the continent. During the period between the 1950s and now been determined at high resolution and is reported by the 1980s, numerous young biologists earned their spend- Conti et al. in this issue of Structure [13]. As beautifully ing money as firefly collectors, first employed by Professor described in their paper, the enzyme folds into two distinct William D McElroy, then of Johns Hopkins University, domains, a large N-terminal domain comprising residues and later as members of the Sigma Firefly Club. In 1985, 4–436, and a C-terminal domain formed from residues when Marlene DeLuca and her colleagues cloned the 440–544. The structure is shown in Figure 1. The fold cDNA encoding the luciferase of P. pyralis, an alternative assumed by the luciferase polypeptide appears to be source of the enzyme became available, and soon after, in unique. The N-terminal domain consists of a ␤ barrel and laboratories around the world, numerous other organisms two ␤ sheets flanked by ␣ helices which form a five-layered began to emit the characteristic yellow-green lumines- ␣␤␣␤␣ structure. The C-terminal domain, consisting of cence as a consequence of expression of the firefly five ␤ strands and three ␣ helices, is folded into a compact luciferase in their cells. structure that is connected to the N-terminal domain by a disordered loop (connecting residues 435 and 441). There Historical perspective are three other disordered loops not visible in the electron Luciferase is a generic term describing any enzyme that density, one in the C-terminal domain connecting residues catalyzes a reaction yielding visible light. Light emission is 523 and 529, and two in the N-terminal domain (connect- a consequence of formation of a product or intermediate in ing residues 198–204 and residues 355–359). an electronically excited state; return to the ground state occurs via emission of a photon of light. Luciferases are The structure presented is without bound substrates or highly diverse, catalyzing a great variety of reactions and other ligands. Conti et al. have taken advantage of the using widely different substrates [1]. All have in common homology of firefly luciferase with other enzymes that cat- the involvement of oxygen. Luciferases are more different alyze similar reactions [14] to deduce the location of the than, for example, proteases, which all carry out hydrolytic active center (see Fig. 2). It is assumed that regions of chemistry on bonds. Luciferases all emit light, but greatest sequence conservation are most likely to be they do so by very different means. Therefore, the involved in the catalytic mechanism of the enzyme. Based luciferases from different organisms probably evolved on their extensive analysis, it is proposed that the active independently, rather than from a common ancestral center is composed of residues on the surfaces of both enzyme. Bacterial luciferase, the first luciferase to be domains, and that upon binding, the domains cloned and also the first to be structurally characterized, is move together to form the active center. The drawings pre- a flavin that utilizes flavin mononu- sented and the comparisons made with the cleotide (FMN) to activate molecular oxygen, yielding a sequences of the large family of homologous proteins con- flavin C4a peroxide. Reaction of the peroxide with an stitute a compelling argument. For a reac- aliphatic aldehyde substrate yields, ultimately, the car- tion to occur with a high quantum yield, as is the case for boxylic acid and the flavin C4a hydroxide in the first firefly luciferase, it is essential that water be excluded from singlet excited state. Light emission, loss of the C4a the . It would appear that the two-domain struc- hydroxide and dissociation of FMN returns the enzyme to ture presented by Conti et al. could serve such a purpose. 224 Structure 1996, Vol 4 No 3

Figure 1

Two orthogonal stereoviews of the surface of firefly luciferase, depicting an ‘anvil and hammer’ motif. Both views show the large N-terminal ‘anvil’ domain below the smaller C-terminal ‘hammer’ domain. The two views are the front view (top) and the right side view (bottom), obtained by rotating the front view through 90° to the left about the vertical axis. The color coding is the same as that used by Conti et al. [13], and indicates the domains and subdomains. The C-terminal domain (yellow) is the only domain composed exclusively of residues that are contiguous in the amino acid sequence, residues 440–544. The three subdomains of the N-terminal anvil consist of stretches that are not contiguous within the overall sequence. Subdomain A (blue) consists of residues 77–222 and 399–405. Subdomain B (purple) consists of residues 22–70 and 236–351. Subdomain C (green) consists of residues 4–10, 363–393 and 418–434. It appears that the active center comprises residues between the anvil and hammer, and it is suggested that the active center forms by movement of these two domains together following substrate binding [13]. (Figure courtesy of Peter Brick.)

The structure of bacterial luciferase has also been deter- appears reasonable to propose that both luciferases exclude mined to high resolution without bound ligands [15]. Bac- water from their respective excited emitters by a confor- terial luciferase is a heterodimer composed of homologous mational rearrangement that occurs upon or following subunits, ␣ and ␤ (see [16] for a review of the bacterial substrate binding. luciferase literature). Both subunits assume the well- known (␤/␣)8, or TIM barrel structure, and are packed Reaction catalyzed by firefly luciferase together by a parallel four-helix bundle. The active center Firefly luciferase catalyzes a multistep reaction [21]. In is confined to the ␣ subunit, and it has been proposed to the first step, luciferin (compound I; Fig. 3) reacts with reside within a large internal cavity which opens through a Mg2+-ATP to form luciferyl adenylate (compound II) and narrow crevice [17]. The opening to the cavity lies pyrophosphate. The luciferyl adenylate is oxidized by beneath a long disordered loop which appears to undergo molecular oxygen, with the intermediate formation of the a conformational rearrangement upon flavin binding and cyclic peroxide, a dioxetanone (compound III), and a mol- [16–20]. It has been proposed that this conforma- ecule of AMP. The dioxetanone is decarboxylated as a tional change may be necessary to exclude water from the result of intramolecular conversions (compound IV) to intermediates and the excited state of the flavin. Thus, it produce an electronically excited state of oxyluciferin in Minireview Luciferases Baldwin 225

Figure 2

Alignment of the amino acid sequences, reported by Devine et al. [49], luciferase from P. pyralis. The extent of conservation at each position is of the luciferases of Photinus pyralis (P.p), Luciola mingrelica (L.m), indicated by the following color code: red=fully conserved in all six L. cruciata (L.c), L. lateralis (L.l), and the green-emitting strain of the sequences; pink=2 different amino acids; green=3 different amino click (CbG). Also shown in this alignment is the acids, and blue=4–6 different amino acids. At positions where a 4-coumarate:CoA (CoA). References to the sequences are deletion has occurred in one or more of the proteins, the given in [49]. The numbering at the top refers to the sequence of the corresponding residues in the other proteins are shown in black.

the or keto form (compound V). Return to the ground cruciata and Luciola lateralis [24], have been purified and state is accompanied by emission of a quantum of visible characterized. The L. mingrelica and L. cruciata luciferases light with a wavelength of maximum light intensity (Imax) are similar to the P. pyralis luciferase, with Imax of of 562–570 nm. Shimomura et al. [22] demonstrated that 562–570 nm, whereas the reaction catalyzed by L. lateralis one oxygen atom of the product CO2 arises from the sub- luciferase emits green light (Imax 552 nm). All Luciola strate oxygen. Non-enzymatic oxidation of luciferin yields luciferases inactivate rapidly at low ionic strength [21], oxyluciferin without luminescence [23]. (For a recent whereas the P. pyralis luciferase crystallizes in active form review, see [12].) under the same conditions [4].

Color of the bioluminescence of The emission spectra of a variety of One of the most intriguing aspects of firefly biolumines- luciferin analogs were studied by White et al. [9,23]. Based cence pertains to the color of the light emitted. Recently, on the emission spectra of these compounds the luciferase from Luciola mingrelica fireflies, collected in in the presence and absence of proton acceptors, they the southern regions of Russia [21], as well as the suggested that the red emission arises from the keto anion luciferases of the fireflies indigenous to Japan, Luciola form of the product molecule and the yellow-green 226 Structure 1996, Vol 4 No 3

Figure 3

Proposed mechanism of the firefly bioluminescence reaction. The carboxylate group of (I) is activated by reaction with ATP to form the adenylated luciferin (II). The ␣ proton is lost, allowing reaction with molecular oxygen to yield (III). Cleavage of the dioxetanone ring (IV) yields the excited state of oxyluciferin (V).

emission from the enol anion. This hypothesis is con- the fact that a large (50 nm) red shift (to an Imax of 612 nm) sistent with the emission of red light in the biolumi- was caused by a single substitution of an invariant his- nescence reaction at lower pH, and has led to the tidinyl residue (conserved in all insect luciferases) by suggestion that the various colors of light observed from a tyrosine (His433→Tyr). When the sequences of the different species of firefly result from a spectral mixing mutant L. cruciata luciferases were compared with those of from these two emitters [5]. However, when luciferases the click beetle enzymes, no common amino acid were isolated from different organs of the click beetle sequence affecting the color of light was apparent [26]. It Pyrophorus plagiophthalamus, each emitted a different color may be concluded that the different colors of biolumines- of bioluminescence, and each emission spectrum showed cence are caused by subtle changes in the tertiary struc- a single peak, not a superimposition of two or more ture of the luciferase molecule. different spectra [24,25]. The cDNAs of four luciferases from P. plagiophthalamus, chosen on the basis of differ- The firefly luciferase superfamily of enzymes ences in color of bioluminescence, have been cloned and The number of proteins related to firefly luciferase and sequenced [26]. The amino acid sequences of these apparently descended from a common ancestor is growing luciferases are 95–99% identical, and fewer than two or rapidly. These enzymes are involved in the biosynthesis of three amino acid changes are needed for spectral shifts of iron-binding siderophores [29,30], the antibiotics grami- up to 50 nm in Imax. The properties of these luciferases cidin S [31,32], tyrocidine [33], and penicillin [34–36], and require some rethinking of the traditional explanations for pathogen defense agents in plants [37,38] and the bio- spectral differences between insect luciferases, which degradation of halogenated aromatics [39]. :coen- historically had been attributed to shifts in the proposed zyme A (CoA) ligase [40] and acetate:CoA ligase [41] use equilibrium distribution between the keto and enol forms the same mechanism as 4-coumarate:CoA ligase [37] and of the excited-state oxyluciferin product. If this were the the proteins show a high degree of sequence similarity. sole explanation for the spectral variations, one would The amino acid sequence of AngR, a DNA-binding protein expect to see intermediate bimodal spectra, as have been that modulates iron-regulated , also shows reported for P. pyralis luciferase titrated with zinc ions homology to the firefly luciferases [42]. Given the diversity [24]. However, the four recombinant click beetle and importance of the related proteins already discovered, luciferases emit light with sharp emission spectra with more are likely to be found as the number of sequenced spectral maxima of 546 nm (green), 560 nm (yellow-green), proteins increases. Each of the enzymes catalyzes the 578 nm (yellow), and 593 nm (orange) [27]. This finding adenylation of a substrate using Mg2+-ATP suggests that light emission from each enzyme occurs (Fig. 4), followed by reaction of the activated carboxylate from a single molecular species within the environment of with an acceptor and release of AMP. Several of the related the enzyme and that differences in color result from enzymes use CoA or an active-site thiol as an acceptor for differences between the microenvironments of the displacing the AMP. There does not appear to be an essen- enzyme–oxyluciferin complexes. In a related study, L. cru- tial thiol in the active site of firefly luciferase [43], although ciata luciferase cDNA was mutagenized and five mutants there have been several reports that the addition of CoA with different colors of bioluminescence ranging from enhances the emission of light from firefly luciferase green to red were isolated [28]. The mutations were found [43–45]. The role, if any, that CoA plays in the firefly to be single amino acid changes. Of particular interest is luciferase reaction remains to be elucidated. Minireview Luciferases Baldwin 227

Figure 4 non-fluorescent flavoprotein (see [16] and other refer- ences therein) — the function of which is unknown.

Even as our knowledge of the structures and mechanisms of these delightful enzymes becomes more detailed, the beauty of the biological phenomenon of bioluminescence will not be diminished. The phenomenon of a flashing firefly will always be a source of joy to those who pause to observe it. The mysterious emission of light without heat will always intrigue the curious; and for the technically more sophisticated, the questions regarding the color of bioluminescence, and the details of the enzyme-catalyzed reaction remain to be resolved. Knowledge of the structure is a major step forward but there is still much to be learned.

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